Our long-range goal is to understand the roles of membrane-bound and cytosolic proteins that direct the sorting of cargo into COPII vesicles budding from endoplasmic reticulum (ER) of mammalian cells. The mechanism by which cargo is selected and concentrated during export is unknown. To address this goal, we will focus on the central role of the Sar1 GTPase and its upstream and downstream effectors in cargo selection. Aim I. Molecular basis for Sar1 activation leading to transitional element formation. We will examine the mechanism by which Sarl directs the assembly of export sites through interaction with the mammalian Sec12 guanine nucleotide exchange factor (GEF) to generate tubular elements that form using a KIF-dependent process. Specific Aim II. Biochemical basis for cargo selection. We will explore the hypothesis that novel components of pre budding complexes are part of a protein machine that coordinates cargo selection with other aspects of ER function to select cargo into COPII vesicles. We will address the molecular basis for cargo recognition, Sec23/24 recruitment and coat polymerization by Sar1, and generate transgenic mouse models to pursue the unique functions of mammalian Sec24 (A-D) isoforms. Specific Aim Ill. Vesicle maturation. We will explore the basis for coordination of cargo recruitment with the recruitment of molecular tethers and targeting/fusion determinants. We will determine the structural organization of the tether p115 using x-ray crystallography. We will explore the contribution of Sec13/31 to the completion of budding and fission of COPII vesicles from the ER through biochemical, molecular and structural analyses, allowing us to test different models for COPII coat polymerization. The proposed studies should allow us to develop significant mechanistic insight into the crucial early events in the secretory pathway that control the flow of nearly one-third of genome into the cell.